Appendix 232 Summary 4H leukodystrophy is a rare genetic disorder characterized by hypomyelination, hypodontia, and hypogonadotropic hypogonadism. Following the discovery that mutations in the genes encoding for RNA polymerase III (Pol III) subunits, such as POLR3A and POLR3B, cause the disorder, clinical diagnosis improved. But, today treatments are still solely symptomatic rather than curative. In this thesis we aimed to gain insight into how molecular and cellular mechanisms contribute to the pathology of 4H leukodystrophy using patientspecific iPSC-based models with the goal to facilitate development of new therapeutic strategies. The first chapter, the introduction, outlines the current state of knowledge on 4H syndrome from clinical presentation to the function of RNA pol III and pathogenic mutations in genes encoding for RNA pol III. We point out that the current treatment options only include symptom relief. Furthermore it presents the rationale for using human iPSC-based models to investigate 4H hypomyelination and evaluate which in vitro models are potentially suitable. In Chapter 2: Cortical interneuron development is affected in 4H leukodystrophy we explored differential expression of genes in 4H cerebellar cells using patient iPSC technology in combination with RNAseq analysis. This revealed downregulation of ARX, a transcription factor required for interneuron development. This was followed by a focused investigation of cortical neuron co-cultures, that showed a reduction in GABAergic synapses and elevated network activity in 4H neurons. Pharmacological manipulation of GABAergic signalling confirmed an inhibitory deficit. Interestingly, myelination appeared normal, and treatment with the Sonic Hedgehog (Shh) agonist SAG did not rescue the phenotype. Expression of the parvalbumin-related gene ERBB4 was increased, suggesting disrupted development of specific interneuron subtypes. Taken together, we concluded that cortical interneuron development is affected in 4H leukodystrophy. In Chapter 3: Investigating neuron intrinsic defects in 4H and Globoid Leukodystrophy we further explored neuronal phenotypes in 4H leukodystrophy. Additionally, we aimed to explore whether neuronal changes are 4H-specific or also occur in other leukodystrophies. Hence control, 4H and GLD neurons were compared. Surprisingly, the in vitro cultures with GLD neurons did not show accumulation of psychosine, a hallmark of the disease. This highlights the heterogeneity of leukodystrophies and the necessity of refining disease
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